CN111699188A - [1,2,4] triazolo [4,3-a ] pyrazin-8-one derivatives - Google Patents

Abstract

The present invention provides compounds of formula I, or pharmaceutically acceptable salts thereof, useful as PDE1 inhibitors:

wherein R is¹Is methyl, ethyl or cyclopropyl; r²Is hydrogen, methyl or ethyl; r³Is methyl or AA

(ii) a And R is⁴Is C2-C4 alkyl, BB

Description

[1,2,4] triazolo [4,3-a ] pyrazin-8-one derivatives

The present invention relates to certain human PDE1 inhibitors, to pharmaceutical compositions comprising said compounds, to methods of using said compounds for the treatment of physiological disorders and to intermediates and processes useful in the synthesis of said compounds.

Phosphodiesterases (PDEs) are enzymes that regulate the cellular levels of these cyclic nucleotides by controlling the rate of hydrolysis of cAMP and cGMP. PDE1 is a calcium and calmodulin-dependent PDE and is one of at least 11 known PDE families. PDE1 is expressed in many tissues, including the brain, heart, lung, kidney, and smooth muscle. PDE1 consists of three known isoforms PDE1A, PDE1B and PDE 1C.

Patients with diabetes often develop some form of chronic kidney disease, known as diabetic kidney disease (or diabetic nephropathy). It has been estimated that diabetic kidney disease may affect up to 40% of diabetic patients. Treatment options for diabetic kidney disease are limited and include the use of blood pressure lowering medications, management of blood glucose levels, diet and weight, and regular physical activity. Thus, patients with chronic kidney disease, particularly diabetic kidney disease, require additional treatment options.

U.S. patent application publication No. 2017/0233396 a1 discloses certain [1,2,4] triazolo [4,3-a ] quinoxalin-4-ones and their use in treating certain diseases, such as chronic kidney disease and diabetic kidney disease. WO 2016/055618 a1 discloses certain triazolopyrazinones as PDE1 inhibitors and their use for the treatment of neurodegenerative and psychiatric disorders.

The present invention provides certain novel compounds which are inhibitors of human PDE 1. The present invention provides certain novel compounds which are selective inhibitors of human PDE1A, PDE1B and PDE1C relative to other human PDE's (e.g., PDE3A, PDE4D and PDE6 AB).

Accordingly, the present invention provides a compound of formula I:

formula I

Wherein R is¹Is methyl, ethyl or cyclopropyl;

R²is hydrogen, methyl or ethyl;

R³is methyl or

(ii) a And is

R⁴Is a C2-C4 alkyl group,

or

。

The present invention also provides a method of treating chronic kidney disease in a patient comprising administering to a patient in need of such treatment an effective amount of a compound of formula I. The present invention also provides a method of treating diabetic kidney disease in a patient comprising administering to a patient in need of such treatment an effective amount of a compound of formula I. The present invention also provides a method of treating hypertension in a patient comprising administering to a patient in need of such treatment an effective amount of a compound of formula I. The present invention also provides a method of treating heart failure in a patient comprising administering to a patient in need of such treatment an effective amount of a compound of formula I.

In addition, the present invention provides compounds of formula I for use in therapy. The invention also provides compounds of formula I for use in the treatment of chronic kidney disease. In addition, the present invention provides compounds of formula I for use in the treatment of diabetic kidney disease. Furthermore, the present invention provides compounds of formula I for use in the treatment of hypertension. The present invention also provides compounds of formula I for use in the treatment of heart failure. The invention also provides the use of a compound of formula I for the preparation of a medicament for the treatment of chronic kidney disease. The invention also provides the use of a compound of formula I for the preparation of a medicament for the treatment of diabetic kidney disease. The invention also provides the use of a compound of formula I for the manufacture of a medicament for the treatment of hypertension. The invention also provides the use of a compound of formula I for the manufacture of a medicament for the treatment of heart failure.

The present invention further provides pharmaceutical compositions comprising a compound of formula I in combination with one or more pharmaceutically acceptable carriers, diluents or excipients. The invention further provides a process for preparing a pharmaceutical composition comprising admixing a compound of formula I with one or more pharmaceutically acceptable carriers, diluents or excipients. The invention also comprises novel intermediates and processes for the synthesis of compounds of formula I.

The term "treating" as used herein includes inhibiting, suppressing, slowing, stopping or reversing the progression or severity of an existing symptom or disorder.

The term "patient" as used herein refers to a mammal, such as a dog or a human, with a human being preferred.

The term "effective amount" as used herein refers to an amount or dose of a compound of the present invention or a pharmaceutically acceptable salt thereof that provides the desired effect in the patient being diagnosed or treated when administered to the patient in a single or multiple dose.

The term "C2-C4 alkyl" as used herein refers to straight, branched and cyclic alkyl groups selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl and cyclobutyl, of which ethyl, n-propyl, cyclopropyl, n-butyl and cyclobutyl are preferred.

The effective amount is readily determined by one skilled in the art by using known techniques and by observing results obtained under similar circumstances. In determining an effective amount for a patient, one skilled in the art takes into account a number of factors, including but not limited to: the size, age, and general health of the patient; the particular disease or condition involved; the degree of involvement or severity of the disease or disorder; the response of the individual patient; the particular compound administered; a mode of administration; the bioavailability characteristics of the administered formulation; a selected dosing regimen; concomitant medication; and other related circumstances.

The compounds of the present invention are effective at daily dosages falling within the range of about 0.01 to about 20 mg/kg body weight. In some cases dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases even larger doses may be employed with acceptable side effects, and therefore the aforesaid dosage range is not intended to limit the scope of the invention in any way.

The compounds of the present invention are formulated into pharmaceutical compositions for administration by any route that makes the compounds bioavailable, including oral and parenteral routes. Such compositions are most preferably for oral administration. Such Pharmaceutical compositions and methods of making them are well known in The art (see, e.g., Remington: The Science and Practice of Pharmacy, L.V. Allen, 22 nd edition, Pharmaceutical Press, 2012).

The compounds of formula I are particularly useful in the methods of treatment of the present invention, but certain groups, substituents and compounds are preferred. The following paragraphs describe these preferred groups, substituents, and compounds. It is to be understood that these preferences apply both to the therapeutic methods of the present invention and to the novel compounds of the present invention.

Preferably, R is¹Is cyclopropyl.

Preferably, R is²Is methyl.

Preferably, R is³Is methyl.

It is further preferred when R is¹When it is cyclopropyl, R²Is methyl.

It is further preferred when R is²When it is methyl, R³Is methyl.

More preferably, when R is¹When it is cyclopropyl, R²And R³Is methyl.

The following compounds and their pharmaceutically acceptable salts are particularly preferred:

and

the corresponding free base of each compound is most particularly preferred.

Pharmaceutically acceptable salts of the compounds of the invention may be formed, for example, by reacting an appropriate free base of a compound of the invention with an appropriate pharmaceutically acceptable acid in an appropriate solvent under standard conditions well known in the art. See, e.g., Gould, P.L., "Salt selection for basic drugs"International Journal of Pharmaceutics33:201-217 (1986); bastin, R.J. et al, "Salt Selection and Optimization procedure for Pharmaceutical New Chemical Entites"Organic Process Research and Development427-435 (2000); and Berge, S.M. et al, "Pharmaceutical Salts"Journal of Pharmaceutical Sciences, 66: 1-19, (1977)。

The individual isomers, enantiomers and diastereomers can be separated or resolved by methods such as selective crystallization techniques or chiral chromatography at any convenient point in the synthesis of the compounds of the invention by one of ordinary skill in the art (see, e.g., j. Jacques et al "Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc., 1981 and E.L. Eliel and S.H. Wilen"Stereochemistry of Organic Compounds", Wiley-Interscience, 1994). The designations "isomer 1" and "isomer 2" refer to the compounds that elute first and second by chiral chromatography, respectively, under specified conditions.

Certain abbreviations are defined as follows: "ACN" refers to acetonitrile; "BSA" refers to bovine serum albumin; "cAMP" refers to cyclic adenosine-3 ',5' -monophosphate; "CDI" refers to 1,1' -carbonyldiimidazole; "cGMP" refers to cyclic guanosine monophosphate; "DCC" refers to 1, 3-dicyclohexylcarbodiimide; "DCM" means dichloromethane or methylene chloride; "DIC" refers to 1, 3-diisopropylcarbodiimide; "DIPEA" refers to N, N-diisopropylethylamine; "DMF" refers to N, N-dimethylformamide; ' DMAP "refers to dimethylaminopyridine; "DMSO" refers to dimethylsulfoxide; "EDCI" refers to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; "EDTA" means ethylenediaminetetraacetic acid; "ES/MS" refers to electrospray mass spectrometry; "EtOAc" refers to ethyl acetate; "Et₂O "means diethyl ether; "EtOH" refers to ethanol or ethyl alcohol; "HATU" refers to 1- [ bis (dimethylamino) methylene]-1H-1,2, 3-triazolo [4,5-b]Pyridinium 3-oxide hexafluorophosphate; "HBTU" means (1H-benzotriazol-1-yloxy) (dimethylamino) -N, N-dimethylmethylidene ammonium (methanoiminium) hexafluorophosphate; "HIS" refers to histidine; "HOAt" refers to 1-hydroxy-7-azobenzotriazole; "HOBt" refers to 1-hydroxybenzotriazole hydrate; ' IC₅₀"refers to the concentration of an agent that produces 50% of the maximum inhibitory response possible for that agent; "MeOH" refers to methanol or methyl alcohol; "MTBE" means methyl-tert-butyl ether; "NiNTA" refers to chromatography on an agarose stationary phase functionalized with nitrilotriacetic acid as a chelating agent; "PDE" refers to phosphodiesterase; "PyBOP" means (benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate); "PyBrOP" refers to bromo (trispyrrolidinyl) phosphonium hexafluorophosphate; "t" s_(R)"refers to retention time; "SFC" refers to supercritical fluid chromatography; "SPA" refers to scintillation proximity assays; "TEA" refers to triethylamine; "THF" refers to tetrahydrofuran; "Tris" means 2-amino-2-hydroxymethyl-propane-1, 3-diol; "U/ml" means units per milliliter; "XPhos Pd G2" refers to chloro (2-dicyclohexylphosphino-2 ', 4', 6 '-triisopropyl-1, 1' -biphenyl) [2- (2 '-amino-1, 1' -biphenyl ]]Palladium (II); and "ee" refers to enantiomeric excess.

The compounds of the present invention may be prepared by a variety of procedures known to those of ordinary skill in the art, some of which are exemplified in the schemes, preparations, and examples below. One of ordinary skill in the art will recognize that the specific synthetic steps of the routes described may be combined in different ways, or with steps from different schemes, to prepare compounds of the invention. The products of the following steps may be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration and crystallization. In the following schemes, all substituents are as defined above unless otherwise indicated. Reagents and starting materials are readily available to those of ordinary skill in the art. The following schemes, preparations and examples are provided to further illustrate the invention without limiting the scope of the invention.

Scheme 1

Scheme 1 depicts alkylation of amine (1) with a substituted nitrile to produce compound (2) as shown in step 1. The alkylation can be achieved using trimethylsilyl cyanide as the nitrile source along with the aldehyde in a solvent such as 1, 2-dimethoxyethane and heating to about 70 ℃. The product can be treated with an acid such as HCl to isolate compound (2) as an acid salt. Alternatively, alkylation can be accomplished using a source of hydroxynitrile and stirred at room temperature in a solvent such as THF and treated with an acid such as HCl to yield compound (2). In step 2, compound (2) may be treated dropwise with an acid chloride, such as oxalyl chloride, at about 0 ℃, followed by heating the reaction to 50-100 ℃ to cyclize the nitrile to the 1, 6-substituted-3, 5-dichloro-pyrazin-2-one, compound (3). In step 3, compound (3) can then be converted to the hydrazide with hydrazine monohydrate in a solvent such as THF at room temperature or in EtOH while heating to about 100 ℃ to yield the corresponding compound (4). In step 4, a suitable carboxylic acid, an organic base such as DIPEA in a solvent such as DMF or DCM and a coupling agent such as N- [ (5-chloro-3-oxoanion (oxido) -1H-benzotriazol-1-yl) -4-morpholinomethylene]-N-methylmethanamine hexafluorophosphate salt to effect amide coupling on compound (4) to produce compound (5). One skilled in the art will recognize that there are many methods and reagents for forming amides from the reaction of carboxylic acids and amines. For example, reaction of an amine compound with an appropriate carboxylic acid in the presence of a coupling reagent, in the presence or absence of an organic base such as DIPEA or TEA, can provide the compound of step 4. Coupling reagents include carbodiimides such as DCC, DIC, EDCI or carbonyl diimidazoles such as CDI. Amide coupling additives such as HOBt and HOAt may also be used to enhance the reaction. In addition, non-nucleophilic anions may be usedSuch as HBTU, HATU, PyBOP and PyBrOP, in place of the more traditional coupling reagents. Additives such as DMAP may be used to enhance the reaction. Alternatively, compound (4) can be acylated in the presence of a base such as TEA or pyridine using the appropriate acid chloride to give compound (5). In step 5, compound (5) may be cyclized to triazole (6) under basic or acidic conditions. For example, treatment of compound (5) with a base such as TEA and thionyl chloride in a solvent such as 1, 4-dioxane in a closed system and heating to about 80 deg.C can provide triazole (6). Alternatively, hexamethyldisilazane may be used as the base and the reaction may be heated to about 120 ℃. After cooling to room temperature, MeOH may be added to promote cyclization. Alternatively, compound (5) may be cyclized to triazole under acidic conditions using an acid such as acetic acid at a temperature of about 130 ℃ under microwave conditions to produce triazole (6). In step 6, two reactions can be achieved. R capable of substituting 5-chloro substituent of Compound (6) to generate hydrogen²Substituents and R may be reacted under Suzuki palladium cross-coupling conditions with a base such as potassium carbonate, a suitable boron reagent (boronicreagent) and a palladium catalyst such as bis (di-tert-butylphosphino) ferrocene palladium dichloride³Further functionalization. The reaction may be heated in a solvent such as DMF at a temperature of about 120 ℃ to yield the compound of formula I. The skilled artisan will recognize that there are a variety of conditions that may be used to facilitate such cross-coupling reactions. Suitable palladium reagents include XPhos Pd Gen 2, bis (triphenylphosphine) palladium (II) chloride, tris (dibenzylideneacetone) dipalladium (0) containing tricyclohexylphosphine, (1,1' -bis (diphenylphosphino) ferrocene) palladium (II) chloride, tetratriphenylphosphine palladium or palladium (II) acetate. Suitable bases include cesium carbonate, sodium carbonate, potassium carbonate, lithium tert-butoxide, or tripotassium phosphate monohydrate.

Scheme 2

In scheme 2, step 1, the 3-chloro substituent of compound (3) as prepared in scheme 1 can be replaced with a methoxy group using sodium methoxide in a solvent such as MeOH at about 0 ℃ to yield compound (7). In step 2, it may then be neutralized in a solvent such as hexaneHeating to about 80 deg.C while in a Negishi palladium cross-coupling reaction with a catalyst such as [1, 3-bis (diphenylphosphino) propane ]]Nickel (II) dichloride and a suitable organozinc reagent functionalize the 5-chloro substituent of Compound (7) to R²To give compound (8). Those skilled in the art are familiar with Negishi couplings involving transition metal catalyzed cross-couplings. This reaction couples the organohalide or triflate with the organozinc compound to form a carbon-carbon bond. Palladium (0) species are often used as metal catalysts, but nickel catalysts may also be used as described above. In step 3, the hydroxyl group can be deprotected using a strong lewis acid such as boron tribromide to yield the 2, 3-dione of compound (9). In step 4, the ketone at the 2-position can be selectively chlorinated in a catalytic amount of DMF using a chlorine source such as thionyl chloride and oxalyl chloride to yield compound (10). In step 5, the chlorine substituent on compound (10) may then be displaced with an appropriate carbohydrazide in a solvent such as THF while heating to about 100 deg.C to yield compound (11). In step 6, compound (11) may be subsequently cyclized as described in scheme 1, step 5 to yield the compound of formula I.

Scheme 3

In scheme 3, step 1, the 2-chloro substituent substituted for 2, 3-dichloropyrazine (12) is converted to compound (13) in a manner analogous to scheme 1, step 3. In scheme 3, step 2, compound (13) is converted to compound (14) in a manner similar to scheme 1, step 4 by amide coupling of a coupling agent such as HATU using a base such as DIPEA in a solvent such as DCM. In scheme 3, step 3, compound (14) may be cyclized in a manner analogous to scheme 1, step 5 to yield compound (15). In scheme 3, step 4, R with a strong non-nucleophilic base such as lithium bis (trimethylsilyl) amide in a solvent such as DMF and potassium iodide acting as a nucleophilic catalyst⁴-the halide alkylates the nitrogen of the pyrazinamide compound (15) to yield the compound of formula I. Alternatively, the lithium bis (trimethylsilyl) amide can be replaced with other bases, such as cesium carbonate or sodium hydride and the mixtureStirring may be carried out at room temperature or heating may be carried out at about 60-80 ℃. DMSO may act as another solvent and a nucleophilic catalyst may not be necessary for a successful reaction.

Preparation 1

2- (cyclopropylmethylamino) propionitrile hydrochloride

Scheme 1, step 1: acetaldehyde (7.89 g, 179.1 mmol) was slowly added to a solution of cyclopropanemethylamine (10.00 g, 137.7 mmol) in 1, 2-dimethoxyethane (78.40 mL, 756.4 mmol) at 0 ℃ and stirred at room temperature for 30 min, followed by dropwise addition of trimethylcyanosilane (20.29 mL,151.5 mmol). The resulting reaction mixture was heated at 70 ℃ for 4 hours and cooled at room temperature. The reaction was cooled to 0 ℃ and kept at N₂HCl (37.893 mL,151.570 mmol) was added dropwise under an atmosphere. The resulting precipitate was filtered and washed with diethyl ether (200 mL) to give the title compound (22.31 g, 97%).

Preparation 2

2- (butylamino) propionitrile; hydrochloride salt

Scheme 1, step 1: a solution of butylamine (6.77 mL, 68.4 mmol) and 2-hydroxypropionitrile (7.39 mL, 103 mmol) in THF (68.4 mL) was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and taken up in Et₂O dilution and dropwise addition of HCl (68 mL, 1.0 mol/L in Et₂In O). The solid formed was collected by filtration to give the title compound (9.67 g, 86.9%).

Preparation 3

1-cyclopropylcyclopropanecarbohydrazide hydrochloride

To a stirred solution of 1-cyclopropylcyclopropanecarboxylic acid (9.63 g, 76.3 mmol) and HATU (32.3 g, 83.2 mmol) in DMF (300 mL) was added tert-butyl carbazate (5.00 g, 37.8 mmol) followed by DIPEA (14.5 mL,83.1 mmol) and the reaction was stirred at room temperature for 5 days. The reaction mixture was diluted with EtOAc and diluted with 1.0N HCl, saturated NaHCO₃And water washing. The organic layer was separated, dried over magnesium sulfate, filtered and concentrated under reduced pressure. 1, 4-dioxane (50 mL) was added to the residue, HCl (4 mol/L) in 1, 4-dioxane (100 mL, 400 mmol) was added over 20 minutes and the reaction was stirred at room temperature for 1 hour. The solution was filtered and the filter cake was washed with MTBE and dried under reduced pressure to give the title compound (8.01 g, 58.7%). MS (M/z) 141 (M + H).

Preparation 4

Methanesulfonic acid (1-methylcyclopropyl) methyl ester

A stirred solution of (1-methylcyclopropyl) methanol (500 mg, 5.805 mmol) and TEA (0.89 mL, 6.39 mmol) in DCM (30 mL) was cooled to 0 ℃ in an ice/water bath. Methanesulfonyl chloride (0.5 mL, 6.46 mmol) was added dropwise via syringe. The reaction mixture was allowed to warm to room temperature; then stirred for 1 hour. Saturated NaHCO was used for the reaction₃Diluted and extracted with DCM. The organics were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (1.00 g, 94%).

Preparation 5

3, 5-dichloro-1- (cyclopropylmethyl) -6-methyl-pyrazin-2-one

Scheme 1, step 2: reacting 2- (cyclopropylmethylamino) propionitrile; a solution of the hydrochloride salt (22.31 g, 134.71 mmol) in 1, 2-dimethoxyethane (216.41 mL; 2.09 mol) was cooled to 0 deg.C and under N₂Oxalyl chloride (23.37 mL, 269.42 mmol) was added dropwise under an atmosphere. The reaction mixture was then brought to room temperature and heated at 100 ℃ for 6 hours. The reaction was cooled to room temperature and stirred overnight. Excess oxalyl chloride was removed under reduced pressure. The mixture was neutralized with saturated bicarbonate solution (100 mL) and extracted with EtOAc (3 × 350 mL). The organic extracts were combined and washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude material. The residue was purified by flash chromatography on silica gel eluted with EtOAc: hexanes to give the title compound (21.33 g, 67.93%). MS (M/z) 235 (M + H).

Preparation 6

1-butyl-3, 5-dichloro-6-methyl-pyrazin-2-one

Scheme 1, step 2: a stirred suspension of 2- (butylamino) propionitrile hydrochloride (9.67 g, 59.4 mmol) in toluene (300 mL) was cooled to 0 ℃ in an ice/water bath. Oxalyl chloride (26.0 mL, 299.7 mmol) was added dropwise. The reaction was stirred at 55 ℃ for 16 h, cooled to room temperature and concentrated under reduced pressure. The residue was purified by flash chromatography on silica, eluting with 0-20% EtOAc/hexanes to give the title compound (14.93 g, > 99%). MS (M/z) 235 (M + H).

Preparation 7

1-butyl-5-chloro-3-methoxy-6-methyl-pyrazin-2-one

Scheme 2, step 1: a stirred solution of 1-butyl-3, 5-dichloro-6-methyl-pyrazin-2-one (12.61 g, 50.42 mmol) in MeOH (15 mL) was cooled to 0 ℃ in an ice/water bath. Sodium methoxide (15 mL, 67 mmol, 25% by mass in MeOH) was added and the mixture was stirred for 20 min. The reaction was diluted with water, and the solid was collected by filtration and dried under reduced pressure to give the title compound (9.39 g, 80.8%). MS (M/z) 231 (M + H).

Preparation 8

1-butyl-5-ethyl-3-methoxy-6-methyl-pyrazin-2-one

Scheme 2, step 2: combine 1-butyl-5-chloro-3-methoxy-6-methyl-pyrazin-2-one (500 mg,2.16 mmol) and [1, 3-bis (diphenylphosphino) propane in a vial]Nickel (II) dichloride (120 mg, 0.221 mmol). In N₂The vial was then sealed, THF (5.5 mL) and diethylzinc solution (6.5 mL, 6.5 mmol,1 mol/L in hexanes) were added and the reaction was stirred overnight at 80 ℃.

Preparation 9

4-butyl-6-ethyl-5-methyl-1H-pyrazine-2, 3-dione

Scheme 2, step 3: a stirred solution of 1-butyl-5-ethyl-3-methoxy-6-methyl-pyrazin-2-one (343.1 mg, 1.53 mmol) in DCM (10 mL) was cooled to 0 ℃ in an ice/water bath. Boron tribromide (3 mL, 3mmol, 1 mol/L in DCM) was added to reactStirred for 2 hours, then warmed to room temperature and stirred for 45 minutes. Saturated NaHCO for reaction₃Quench and extract the aqueous phase with DCM (3 ×). the combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (320.3 mg, 89%). MS (M/z) 211 (M + H).

Preparation 10

1-butyl-3-chloro-5-ethyl-6-methyl-pyrazin-2-one

Scheme 2, step 4: a solution of 4-butyl-6-ethyl-5-methyl-1H-pyrazine-2, 3-dione (301.3 mg, 1.29 mmol), thionyl chloride (1.0 mL, 13.73 mmol) and catalytic DMF (3 drops) in DCM (6 mL) was stirred at room temperature for 45 min. Additional thionyl chloride (1.0 mL, 13.73 mmol) was added and the reaction stirred for an additional 45 minutes. The reaction was concentrated under reduced pressure. The residue was suspended in toluene and concentrated under reduced pressure (2 ×) to give the title compound (475.9 mg, 96.8%,60 mass%). MS (M/z) 229 (M + H).

Preparation 11

5-chloro-1- (cyclopropylmethyl) -3-hydrazino-6-methyl-pyrazin-2-one

Scheme 1, step 3: hydrazine monohydrate (15.3 mL,201.3 mmol) was added dropwise to a solution of 3, 5-dichloro-1- (cyclopropylmethyl) -6-methyl-pyrazin-2-one (21.33 g, 91.51 mmol) in THF (106.7 mL, 1311 mmol), cooled to 0 ℃, stirred for 15 min, then at room temperature for 16 h. Water (100 mL) was added and the mixture was extracted with DCM (300 mL). The organic extracts were concentrated under reduced pressure to give a yellow solid, which was in Et₂Triturated in O (100 mL) and then filtered to give the title compound (0.26 g, 82%). MS (M/z) 229 (M + H).

Preparation 12

N' - [ 6-chloro-4- (cyclopropylmethyl) -5-methyl-3-oxo-piperazin-2-yl ] -1-cyclopropyl-cyclopropanecarbohydrazide

Scheme 1, step 4: in N₂1-Cyclopropylcyclopropanecarboxylic acid (10.13 g,80.28 mmol) was added to a stirred solution of 5-chloro-1- (cyclopropylmethyl) -3-hydrazino-6-methyl-pyrazin-2-one (16.69 g, 72.98 mmol) and DIPEA (42.00 mL, 240.8 mmol) in anhydrous DMF (417.3 mL) at room temperature under an atmosphere followed by N- [ (5-chloro-3-oxoanion-1H-benzotriazol-1-yl) -4-morpholinomethylene-methylene-ene]-N-methylmethanmonium hexafluorophosphate (36.59 g,80.28 mmol). The reaction mixture was stirred at room temperature for 2 hours. Water (1.4L) was added to the reaction mixture and a precipitate formed. The reaction mixture was filtered and the solid isolated using Et₂O (1L) wash to give the title compound (16.06 g, 65%). MS (M/z) 339 (M + H).

Preparation 13

N' - (4-butyl-6-ethyl-5-methyl-3-oxo-piperazin-2-yl) -1-cyclopropyl-cyclopropanecarbohydrazide

Scheme 2, step 5: in N₂1-butyl-3-chloro-5-ethyl-6-methyl-pyrazin-2-one (475.9 mg, 1.25mmol, 60 mass%), 1-cyclopropylcyclopropanecarbohydrazide hydrochloride (221 mg, 1.25 mmol) and THF (4 mL) were combined in a sealed microwave vial and stirred under microwave conditions at 100 ℃ for 2H the reaction was diluted with water and extracted with DCM (3 ×). the combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (320.3 mg, 89%, 70 mass%). MS (M/z) 333 (M + H).

Preparation 14

(3-chloro-5, 6-dimethyl-pyrazin-2-yl) hydrazine

Scheme 3, step 1: a stirred solution of 2, 3-dichloro-5, 6-dimethyl-pyrazine (2.0 g, 11.298 mmol) and hydrazine (0.943 mL,28.2 mmol, 95% by mass) in EtOH (15 mL) was heated at 100 ℃ overnight. The reaction mixture was concentrated under reduced pressure to give the title compound (1.94 g, 84.6%). MS (M/z) 173 (M + H).

Preparation 15

N' - (3-chloro-5, 6-dimethyl-pyrazin-2-yl) -1-cyclopropyl-cyclopropanecarbohydrazide

Scheme 3, step 2: (3-chloro-5, 6-dimethyl-pyrazin-2-yl) hydrazine (4.08 g, 23.6 mmol), 1-cyclopropylcyclopropanecarboxylic acid (4.77 g, 37.8 mmol), HATU (14.7 g, 37.9 mmol) and DIPEA (14.4 mL, 82.6 mmol) were dissolved in DCM (120 mL) and stirred at room temperature for 45 min. The reaction mixture was washed with water, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography on silica, eluting with 0-100% EtOAc/hexanes to give the title compound (4.37 g, 65.8%). MS (M/z) 280 (M + H).

The following compounds are prepared in a manner substantially analogous to the procedure for preparation 15.

TABLE 1

Preparation number	Chemical name	Structure of the product	MS (m/z) (M+H)
				16	N' - (3-chloro-5, 6-dimethyl-pyrazin-2-yl) -1-ethyl-cyclopropanecarbohydrazide		269
17	N' - (3-chloro-5, 6-dimethyl-pyrazin-2-yl) -1-methyl-cyclopropanecarbohydrazide		255

Preparation 18

5-chloro-3- (1-cyclopropylcyclopropyl) -7- (cyclopropylmethyl) -6-methyl- [1,2,4] triazolo [4,3-a ] pyrazin-8-one

Scheme 1, step 5: n' - [ 6-chloro-4- (cyclopropylmethyl) -5-methyl-3-oxo-piperazin-2-yl ] -1-cyclopropyl-cyclopropanecarbohydrazide (18.44 g, 54.75 mmol) was added to 1, 4-dioxane (547.5 mL, 6413 mmol), followed by the addition of TEA (30.52 mL, 219.0 mmol) and thionyl chloride (7.98 mL, 109.5 mmol). The reactor was closed and stirred at room temperature for 30 minutes, then heated at 80 ℃ for 2 hours. The reaction was cooled to room temperature and water (1L) was added. The mixture was extracted with DCM (2 × 750 ml). The organic extracts were combined and dried over sodium sulfate; filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash chromatography on silica gel eluted with EtOAc: hexanes to give the title compound (9.2 g, 52%). MS (M/z) 321 (M + H).

Preparation 19

3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl-7H- [1,2,4] triazolo [4,3-a ] pyrazin-8-one

Scheme 3, step 3: n' - (3-chloro-5, 6-dimethyl-pyrazin-2-yl) -1-cyclopropyl-cyclopropanecarbohydrazide (3.74 g, 7.99 mmol, 60 mass%) was dissolved in acetic acid (15 mL) and heated under microwave irradiation at 130 ℃ for 3 hours. The reaction was cooled to room temperature, the solid was collected by filtration and washed with hexane to give the title compound (1.98 g, 100%). MS (M/z) 245 (M + H).

The following compounds are prepared in a manner substantially analogous to the procedure for preparation 19.

TABLE 2

Preparation number	Chemical name	Structure of the product	MS (m/z) (M+H)
				20	5, 6-dimethyl-3- (1-methylcyclopropyl) -7H- [1,2,4]Triazolo [4,3-a]Pyrazin-8-ones		219
21	3- (1-ethylcyclopropyl) -5, 6-dimethyl-7H- [1,2,4]Triazolo [4,3-a]Pyrazin-8-ones		233

Example 1

3- (1-cyclopropylcyclopropyl) -7- (cyclopropylmethyl) -6- [ (1-methylpyrazol-4-yl) methyl ] - [1,2,4] triazolo [4,3-a ] pyrazin-8-one

Scheme 1, step 6: potassium carbonate (0.127 g, 0.922 mmol) was added to 5-chloro-3- (1-cyclopropylcyclopropyl) -7- (cyclopropylmethyl) -6-methyl- [1,2,4]Triazolo [4,3-a]Pyrazin-8-one (0.098 g, 0.307 mmol) and 1-methyl-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrazole (0.0476 g, 0.229 mmol) in DMF (4.0 mL). The reaction is degassed and treated with N₂Purge for 10 minutes. 1,1' -bis (di-tert-butylphosphino) ferrocene palladium dichloride (0.015 g, 0.022 mmol) was added to the reaction and the mixture was heated at 120 ℃ for 16 hours. The reaction was performed in EtOAc and cold saturated NaHCO₃The solutions were partitioned and separated. The organic layer was washed with 5% lithium chloride (aq), followed by brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 0-100% acetone/hexanes to give the title compound (0.015 mg, 13%). MS (M/z) 365 (M + H).

Example 2

3- (1-cyclopropylcyclopropyl) -7- (cyclopropylmethyl) -5, 6-dimethyl- [1,2,4] triazolo [4,3-a ] pyrazin-8-one

Scheme 3, step 4: lithium bis (trimethylsilyl) amide (2.5 mL, 2.5 mmol,1 mol/L in MTBE) was added to 3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl-7H- [1,2,4] triazolo [4,3-a ] pyrazin-8-one (202 mg, 0.827 mmol) in DMF (8 mL) and the reaction was stirred at room temperature for 1H. (bromomethyl) cyclopropane (400 μ L, 4 mmol) and potassium iodide (15 mg, 0.0909 mmol) were added and the reaction was stirred at room temperature overnight. Additional (bromomethyl) cyclopropane (80 μ L, 0.8 mmol) and potassium iodide (15 mg, 0.0909 mmol) were added and the reaction stirred at room temperature for 4 hours then at 35 ℃ overnight. The reaction was partitioned between EtOAc and water and separated. The aqueous material was extracted with EtOAc. The organic layer was washed with 5% lithium chloride (aq), followed by brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was combined with previous material prepared in essentially the same manner as described in alternative example 2 and purified by flash chromatography on silica gel eluting with 0-10% MeOH in DCM. The isolated material was combined with the previous material prepared in essentially the same manner (51 mg scale reaction) and recrystallized from EtOAc and dried in a vacuum oven to give the title compound (140.3 mg,16% overall yield). MS (M/z) 299 (M + H).

The following compounds are prepared in a manner substantially analogous to the procedure of example 2.

TABLE 3

Example numbering	Chemical name	Structure of the product	MS (m/z) (M+H)
				3	3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl-7-pentyl- [1,2,4]Triazolo [4,3-a]Pyrazin-8-ones		315
4	7- (cyclobutylmethyl) -3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl- [1,2,4]Triazolo [4,3-a]Pyrazin-8-ones		313

Alternative embodiment 2

3- (1-cyclopropylcyclopropyl) -7- (cyclopropylmethyl) -5, 6-dimethyl- [1,2,4] triazolo [4,3-a ] pyrazin-8-one

Scheme 3, step 4: combine 5, 6-dimethyl-3- (1-methylcyclopropyl) -7H- [1,2,4] in DMF (1 mL)]Triazolo [4,3-a]Pyrazin-8-one (25 mg, 0.07 mmol), cesium carbonate (100 mg, 0.31 mmol), potassium iodide (3 mg,0.02 mmol) and bromomethylcyclopropane (25 μ L, 0.26 mmol). Mixture in N₂The reaction was cooled to room temperature and diluted with EtOAc and washed with water (2 ×). the organic layer was washed with 5% lithium chloride (aq), dried over anhydrous sodium sulfate, isolated and concentrated under reduced pressure.

Example 5

7-butyl-3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl- [1,2,4] triazolo [4,3-a ] pyrazin-8-one

Scheme 3, step 4: sodium hydride (950 mg, 23.75 mmol, 60 mass% in mineral oil) was added to 3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl-7H- [1,2,4] triazolo [4,3-a ] pyrazin-8-one (1.95 g, 7.98 mmol) in DMF (50 mL) at 0 ℃. 1-bromobutane (2.15 mL, 19.9 mmol) was added and the reaction was stirred at room temperature overnight. The reaction was then stirred at 60 ℃ for 2 hours. The reaction was cooled to rt and diluted with EtOAc. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica, eluting with 0-50% EtOAc/hexanes, then 0-10% MeOH/DCM. The product-containing chromatographic fractions were combined and concentrated under reduced pressure. The impure residue was purified by flash chromatography on silica eluting in 0-100% EtOAc/DCM. The chromatographic fractions containing the product were combined, concentrated under reduced pressure and lyophilized to give the title compound (100 mg, 4.7%). MS (M/z) 301 (M + H).

Example 6

Racemic 3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl-7- (tetrahydropyran-2-ylmethyl) - [1,2,4] triazolo [4,3-a ] pyrazin-8-one

Scheme 3, step 4: combining 3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl-7H- [1,2,4] in DMF (66 mL)]Triazolo [4,3-a]Pyrazin-8-one (1.95 g, 7.98 mmol), cesium carbonate (7.75 g, 23.8 mmol), potassium iodide (131 mg, 0.789 mmol) and 2- (bromomethyl) tetrahydro-2H-pyran (1.80 mL, 17.8 mmol). Mixture in N₂The mixture was stirred at 80 ℃ for 6 hours. The reaction was cooled to room temperature and diluted with 3:1 chloroform/isopropanol. Saturated NaHCO for organic matter₃Followed by brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is oxidized in the presence of oxygenPurify by flash chromatography on silica eluting with 0-100% EtOAc/hexanes then 0-10% MeOH/DCM to give the title compound (275 mg, 10%). MS (M/z) 343 (M + H).

The following compounds are prepared in a manner substantially analogous to the procedure of example 6.

TABLE 4

Example numbering	Chemical name	Structure of the product	MS (m/z) (M+H)
				7	Racemic 3- (1-ethylcyclopropyl) -5, 6-dimethyl-7- (tetrahydropyran-2-ylmethyl) - [1,2,4]Triazolo [4,3-a]Pyrazin-8-ones		331
8	Racemic 5, 6-dimethyl-3- (1-methylcyclopropyl) -7- (tetrahydropyran-2-ylmethyl) - [1,2,4]Triazolo [4,3-a]Pyrazin-8-ones		317

Example 9

3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl-7- (tetrahydropyran-2-ylmethyl) - [1,2,4] triazolo [4,3-a ] pyrazin-8-one; isomer 1

And

example 10

3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl-7- (tetrahydropyran-2-yl ] methyl ] - [1,2,4] triazolo [4,3-a ] pyrazin-8-one, isomer 2

Racemic 3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl-7- (tetrahydropyran-2-yl)]Methyl radical]-[1,2,4]Triazolo [4,3-a]Pyrazin-8-one (250 mg, 0.730 mmol) was separated into its constituent enantiomers by chiral chromatography using the following conditions: column (S, S) Whelk-0125 cm x 21.2 mm, 10 mu m; 21X 250 mm, mobile phase 35% MeOH 65% CO₂Column temperature 40 ℃, flow rate 5 mL/min, UV 225. The first eluted material was lyophilized to give the title compound of example 9 (95 mg, 38%), MS (M/z) 343 (M + H), t_(R)= 1.81 min, ee>99 percent. The second eluting isomer was lyophilized to give the title compound of example 10 (95 mg, 38%), MS (M/z) 343 (M + H), t_(R)= 2.62 min, ee>99%。

Example 11

3- (1-ethylcyclopropyl) -5, 6-dimethyl-7- (tetrahydropyran-2-ylmethyl) - [1,2,4] triazolo [4,3-a ] pyrazin-8-one, isomer 1

And

example 12

3- (1-ethylcyclopropyl) -5, 6-dimethyl-7- (tetrahydropyran-2-ylmethyl) - [1,2,4] triazolo [4,3-a ] pyrazin-8-one; isomer 2

Racemic 3- (1-ethylcyclopropyl) -5, 6-dimethyl-7- (tetrahydro-cyclopropyl)Pyran-2-ylmethyl) - [1,2,4]Triazolo [4,3-a]Pyrazine-8-one (564 mg, 1.71 mmol) was separated into its component enantiomers by chiral SFC using column (S, S) Whelk-0125 cm × 21.2 mm, 10 μ, mobile phase 40% EtOH 75% CO₂Flow rate 5 mL/min, UV 225 nm, column temperature 35 ℃. The first eluate was isolated as the title compound of example 11 (262 mg, 46.5%), MS (M/z) 331 (M + H), t_(R)= 1.99 min, ee>99 percent. The second eluting material was isolated as the title compound of example 12 (248 mg, 44%), MS (M/z) 331 (M + H), t_(R)= 3.03 min, ee>99%。

Example 13

5, 6-dimethyl-3- (1-methylcyclopropyl) -7- (tetrahydropyran-2-ylmethyl) - [1,2,4] triazolo [4,3-a ] pyrazin-8-one; isomer 1

And

example 14

5, 6-dimethyl-3- (1-methylcyclopropyl) -7- (tetrahydropyran-2-ylmethyl) - [1,2,4] triazolo [4,3-a ] pyrazin-8-one; isomer 2

Racemic 5, 6-dimethyl-3- (1-methylcyclopropyl) -7- (tetrahydropyran-2-ylmethyl) - [1,2,4]Triazolo [4,3-a]Pyrazine-8-one (210 mg, 0.664 mmol) was separated into its component enantiomers by chiral SFC using column (S, S) Whelk-0125 cm × 21.2 mm, 10 μ, mobile phase 35% EtOH/CO₂Flow rate 5 mL/min, UV 225 nm, column temperature 40 ℃. The first eluted material was separated to give the title compound of example 13 (60 mg, 28.6%), MS (M/z) 317 (M + H), t_(R)= 1.84 min, ee>99 percent. The second eluted material was separated to give the title compound of example 14 (55 mg, 26.2%), MS (M/z) 317 (M + H), t_(R)= 2.65 min, ee>99%。

Example 15

3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl-7- [ (1-methylcyclopropyl) methyl ] - [1,2,4] triazolo [4,3-a ] pyrazin-8-one

Scheme 3, step 4: combining 3- (1-cyclopropylcyclopropyl) -5, 6-dimethyl-7H- [1,2,4] in DMSO (7 mL)]Triazolo [4,3-a]Pyrazin-8-one (350 mg, 1.43 mmol), cesium carbonate (1.40 g, 4.3 mmol) and methanesulfonic acid (1-methylcyclopropyl) methyl ester (250 mg, 1.52 mmol). The mixture is in N₂Stirred at room temperature overnight. The reaction was diluted with EtOAc and washed with brine. The aqueous layer was extracted with EtOAc, and the combined organics were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by washing with 10-60% ACN/H on C18₂Purification by reverse phase flash chromatography eluting with O, and lyophilization gave the title compound (2 mg, 0.45%). MS (M/z) 313 (M + H).

Example 16

7-butyl-3- (1-cyclopropylcyclopropyl) -5-ethyl-6-methyl- [1,2,4] triazolo [4,3-a ] pyrazin-8-one

Scheme 2, step 6: n' - (4-butyl-6-ethyl-5-methyl-3-oxo-piperazin-2-yl) -1-cyclopropyl-cyclopropanecarbohydrazide (576.1 mg, 1.213 mmol, 70 mass%) was stirred in hexamethyldisilazane (4 mL) at 120 ℃ overnight. The reaction mixture was cooled to room temperature and poured into MeOH. After addition to MeOH, the reaction mixture erupted vigorously. The resulting residue was purified by flash chromatography on silica gel eluting with 0-10% MeOH/DCM. The resulting residue was dissolved in hexamethyldisilazane (4 mL) and stirred at 120 ℃ overnight. The reaction mixture was cooled to room temperature, MeOH was added, the reaction stirred at 50 ℃ for 30 min and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 0-10% MeOH/DCM. The material was further purified by reverse phase flash chromatography on C18 at 10-100% ACN/H₂O (0.1% ammonium bicarbonate) to give the title compound (101.7 mg, 27%). MS (M/z) 315 (M + H).

Production of PDE proteins

The nucleotide sequences encoding full-length human PDE1A (NP _001003683.1) and PDE1C (NP _005011.1) were inserted into the pFastBac1 (Invitrogen) vector with an N-terminal HIS tag. The nucleotide sequences encoding the catalytic domains (residues 641-1141) of full-length human PDE4D (NP-006194.2) and PDE3A (NP-000912.3) were inserted into the pFastBac1 (Invitrogen) vector with the C-terminal HIS tag. The nucleotide sequences encoding full-length human PDE6A (NP _000431.2) and PDE6B (AAH00249.1) were inserted into pfastbacdual (invitrogen) vectors with an N-terminal HIS tag and an N-terminal Flag tag, respectively, to generate the PDE6A/6B dimer. Baculovirus production and protein expression in Sf9 cells were performed according to the protocol of Bac-to-Bac expression system (Invitrogen). The nucleotide sequence encoding full-length human PDE1B (NP _000915.1) was inserted into pIEX4 (Novagen) with a C-terminal HIS tag and both protein production in Sf9 cells was performed according to the supplier's protocol (Novagen). Using Ni-NTA agarose (Qiagen), followed by SUPERDEX^®Size exclusion chromatography on 200 columns (GE Healthcare) purified His-tagged PDE proteins in storage buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol). Flag-tagged PDE proteins including PDE6A/6B were purified using anti-Flag M2-Sepharose (Sigma) after purification by NiNTA column chromatography and eluted in storage buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1 mg/ml Flag peptide). All purified proteins were stored in small aliquots at-80 ℃.

Phosphodiesterase enzyme detection

All 3',5' cyclic nucleotide PDE enzyme activities were measured using a radioactive enzyme assay based on the SPA detection system. Test compounds were diluted in pure DMSO using a ten-point concentration response curve. The maximum compound concentration in the reaction mixture was 10 or 100 μ M. The compounds were preincubated with either PDE enzyme for 30 minutes at the appropriate concentration, and the reaction was then initiated by addition of substrate. The reaction was allowed to proceed for 60 minutes at room temperature. Next, the reaction was stopped by adding SPA beads. After 12 hours at MICROBETA^TMTRILUX^®The sample is read in a counter. By plotting normalized data vs. log [ compounds ]]And calculating IC using four-parameter logistic equation fitting data₅₀The value is obtained.

Ca²⁺Calmodulin-dependent PDE enzyme assay

PDE1B, PDE1A and PDE1C were cloned and purified according to standard protein production procedures. The detection buffer was prepared to obtain 50 mM Tris-HCl, 50 mM MgCl in water at pH 7.5₂、4 mM CaCl₂Final concentrations in the assay of 0.1% BSA and 6U/ml calmodulin. The final enzyme concentrations were 0.25, 0.074 and 0.0012nM for PDE1A, PDE1B and PDE1C, respectively. By adding the substrate [2 ]³H]cAMP initiates the reaction to give a final concentration of 47 nM.

In vitro potency of the Compounds of the examples on human PDE1A, PDE1B and PDE1C

The data in Table 5 demonstrate that the compounds of examples 1-16 inhibit human PDE1A, PDE1B and PDE1C enzyme activity in vitro.

Use of³H]PDE enzyme detection with cAMP as substrate

Use of³H]cAMP as a reaction substrate the following PDE activities were measured: human PDE3A (catalytic domain) and human PDE 4D. Both enzymes were cloned and purified according to standard procedures. Preparation of assay buffer to obtain 50 mM Tris-HCl, 8.3mM MgCl at pH 7.5₂Final concentrations in the assay of 1.7 mM EDTA and 0.1% BSA. The final enzyme concentrations were 0.008 and 0.021 nM for PDE3A and PDE4D, respectively. By adding the substrate [2 ]³H]cAMP initiates the reaction to give a final concentration of 47 nM.

In vitro potency of the Compounds of the examples on human PDE3A (catalytic Domain) and PDE4D

Use of³H]PDE enzyme assay with cGMP as substrate

Use of³H]cGMP was used as a reaction substrate to measure the following phosphodiesterase activities: human PDE 6A/6B. Catalytically active forms of human PDE6Is a dimer consisting of α (human PDE 6A) and β subunits (human PDE 6B) dimer of human PDE6A/6B was generated by a co-expression and purification strategy (using two purification steps, NiNTA and anti-FLAG Sepharose chromatography). assay buffer was prepared to give 50 mM Tris-HCl, 8.3mM MgCl at pH 7.5₂Final concentrations in the assay of 1.7 mM EDTA and 0.1% BSA. The final enzyme concentration was 5 nM. By adding the substrate [2 ]³H]cGMP initiates the reaction to give a final concentration of 80 nM.

In vitro potency of the compounds of the examples on PDE6AB

The data in tables 5,6 and 7 demonstrate that the compounds of examples 1-16 are selective inhibitors of human PDE1A, PDE1B and PDE1C in vitro relative to human PDE3A, PDE4D and PDE6 AB.

Claims (15)

1. A compound of the formula:

wherein R is¹Is methyl, ethyl or cyclopropyl;

R²is hydrogen, methyl or ethyl;

R³is methyl or

(ii) a And is

R⁴Is a C2-C4 alkyl group,

or

。

2. A compound or salt according to claim 1, wherein R¹Is cyclopropyl.

3. A compound or salt according to claim 1 or claim 2, wherein R²Is methyl.

4. A compound or salt according to any one of claims 1 to 3, wherein R³Is methyl.

5. The compound or salt according to claim 1, wherein the compound is:

。

6. the compound or salt according to claim 1, wherein the compound is:

。

7. the compound or salt according to claim 1, wherein the compound is:

。

8. the compound or salt according to claim 1, wherein the compound is:

。

9. a method of treating chronic kidney disease in a patient, comprising administering to a patient in need thereof an effective amount of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.

10. A method of treating diabetic kidney disease in a patient, comprising administering to a patient in need thereof an effective amount of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.

11. A compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, for use in therapy.

12. A compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, for use in the treatment of chronic kidney disease.

13. A compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, for use in the treatment of diabetic kidney disease.

14. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

15. A process for preparing a pharmaceutical composition comprising mixing a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents or excipients.